高效利用有机碳源蓝细菌新底盘的构建及应用

Cyanobacteria are emerging hosts for sustainable production of biofuels and valuable biochemicals. Even so, the productivity is still too low to meet the demands from industry. Mixotrophy is now being recognized as a favorable strategy for cyanobacteria to have high cell density and product accumulation as cyanobacteria can obtain more extra carbon and energy sources from sugars. However, the conversion efficiency of glucose to the target product is still low, which limits the further application of cyanobacterial chassis under mixotrophic condition. Therefore, we propose: 1) rewiring the central carbon metabolism network by introducing an artificial acetyl-CoA metabolic pathway to minimize unnecessary loss of carbon source and improve the utilization efficiency of glucose in the cyanobacterial chassis; 2) construction of a novel cyanobacterial chassis adapted to mixotrophy by establishing of dynamic regulation modules for endogenous glycolysis pathway and the artificial pathway to balance the carbon flux by identification and construction of the quorum-sensing circuit in cyanobacteria; 3) construction of the heterologous 3-hydroxypropionic acid synthesis pathway in the new chassis and optimization of the culture system under mixotrophy to analyze the conversion efficiency of the glucose towards target chemicals. The successful implementation of this project will be helpful to establish new strategies for rational design cyanobacterial chassis under mixotrophic growth conditions, which will lay groundwork for the industrial application of the "Cyanobacterial Cell Factory" in the future.

近年来，在蓝细菌底盘中已实现多种生物燃料和精细化学品的光合生物合成。但目前的挑战是光合制造的产率还很低，难以满足工业应用的需求。光照条件下添加葡萄糖光混养有助于蓝细菌的高密度培养和产品产量的提高，但葡萄糖转化成目标产品的效率较低，限制了蓝细菌底盘的进一步应用。鉴于此，本项目提出：①引入异源非氧化可循环糖酵解途径组装乙酰辅酶A代谢通路，降低本底糖酵解途径合成乙酰辅酶A过程中碳源的不必要损失，提高蓝细菌底盘对葡萄糖的利用效率；②鉴定适用于蓝细菌底盘的群体感应系统调控元件，建立本底糖酵解途径与人工组装路径的动态调控模块，平衡代谢网络碳通量，构建适配光混养培养的新型蓝细菌底盘；③利用平台化合物三羟基丙酸生物合成系统，建立新底盘的光混养培养体系，检验蓝细菌新底盘中葡萄糖的转化效率。项目将为光混养条件下蓝细菌底盘的理性设计提供新策略，为蓝细菌细胞工厂这一绿色可再生性生物合成体系的工业化应用奠定基础。

蓝细菌是一类能够进行光合作用的原核生物，因其具有生长周期比植物短、便于遗传操作、培养方式简单等优势，已被广泛开发作为细胞工厂用于生物燃料和精细化学品的光合生物合成。尽管目前利用蓝细菌底盘进行生物合成的研究越来越多，但蓝细菌底盘作为细胞工厂进行生物合成的大规模生产并不普遍，目前的困难是光合制造的产率还很低，难以满足工业应用的需求。在光照条件下添加葡萄糖光混养的营养模式，有助于蓝细菌的高密度培养和产品产量的提高，为蓝细菌细胞工厂的大规模应用提供了可行性策略。但光混养模式下，葡萄糖转化成目标产品的效率较低，限制了蓝细菌底盘的进一步应用。考虑到这些问题，本项目通过引入异源非氧化可循环糖酵解途径组装乙酰辅酶A代谢通路，降低本底糖酵解途径合成乙酰辅酶A过程中碳源的不必要损失，通过重新连接蓝细菌中心碳代谢通路，提高葡萄糖的利用效率，增加了关键代谢物乙酰辅酶A的含量。此外，本项目还利用平台化合物三羟基丙酸生物合成系统，建立新底盘的光混养培养体系，检验蓝细菌新底盘中葡萄糖的转化效率。通过项目研究，为光混养条件下蓝细菌底盘的理性设计提供新策略，为蓝细菌细胞工厂这一绿色可再生性生物合成体系的工业化应用奠定基础。